Research obiective no. 14: Functional Senescence. The receptor for advanced glycation endproducts (RAGE) interacts with several functionally and structurally dissimilar ligands. Two of these ligands, advanced glycation endproducts (AGEs) and amyloid fibrils, accumulate with biological aging and during certain disease conditions. Interaction of these ligands with RAGE contributes to disease pathology. While significant research effort has been focused on understanding the pathophysiological effects of RAGE-ligand interactions, little is known about these interactions at a molecular level; how a multiligand receptor like RAGE can recognize structurally unrelated ligands is a key question. A detailed study of RAGE-ligand interactions would benefit understanding of the pathological effects of aging and age-related disorders and may lead to development of therapeutic agents. For the proposed research, an expression system for production of the RAGE extracellular domain in E. coil will be developed. The recombinant receptor will be used to study binding of RAGE ligands, including AGE-modified proteins and amyloid fibrils, prepared in vitro. Changes in the biophysical properties of the protein stubstrates, as they are converted to RAGE ligands, will be examined by a variety of techniques such as fluorescence, circular dichroism, and gel filtration chromatography. The ligand binding site(s) of RAGE will be characterized by screening combinatorial peptide phage display libraries. Conditions will be screened for crystallization of the recombinant RAGE and, if successful, the structure of RAGE extracellular domain will be determined by x-ray crystallography using MAD or SAD techniques.